Multigenic truncation of the semaphorin-plexin pathway by a germline chromothriptic rearrangement associated with Moebius syndrome Nazaryan-Petersen, Lusine; Oliveira, Inês R; Mehrjouy, Mana M; Mendez, Juan M M; Bak, Mads; Bugge, Merete; Kalscheuer, Vera M; Bache, Iben; Hancks, Dustin C; Tommerup, Niels Published in: Human Mutation DOI: 10.1002/humu.23775 Publication date: 2019 Document version Peer reviewed version Citation for published version (APA): Nazaryan-Petersen, L., Oliveira, I. R., Mehrjouy, M. M., Mendez, J. M. M., Bak, M., Bugge, M., Kalscheuer, V. M., Bache, I., Hancks, D. C., & Tommerup, N. (2019). Multigenic truncation of the semaphorin-plexin pathway by a germline chromothriptic rearrangement associated with Moebius syndrome. Human Mutation, 40(8), 1057- 1062. https://doi.org/10.1002/humu.23775 Download date: 27. Sep. 2021 HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Hum Mutat Manuscript Author . Author manuscript; Manuscript Author available in PMC 2020 August 01. Published in final edited form as: Hum Mutat. 2019 August ; 40(8): 1057–1062. doi:10.1002/humu.23775. Multigenic truncation of the semaphorin-plexin pathway by a germline chromothriptic rearrangement associated with Moebius syndrome Lusine Nazaryan-Petersen1,*, Inês R. Oliveira1,2, Mana M. Mehrjouy1, Juan M.M. Mendez1, Mads Bak1,3, Merete Bugge1, Vera M. Kalscheuer4, Iben Bache1,3, Dustin C. Hancks5, Niels Tommerup1,* 1Wilhelm Johannsen Center for Functional Genome Research, Institute of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, 2200, Denmark 2Faculty of Pharmacy, University of Lisbon, Lisbon, 1649-003, Portugal 3Department of Clinical Genetics, Copenhagen University Hospital, Rigshospitalet, Copenhagen, 2100, Denmark 4Group Development and Disease, Max Planck Institute for Molecular Genetics, Berlin, 14195, Germany 5Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, 75235, TX, USA Abstract Moebius syndrome (MBS) is a congenital disorder caused by paralysis of the facial and abducens nerves. While a number of candidate genes have been suspected, so far only mutations in PLXND1 and REV3L are confirmed to cause MBS. Here, we fine mapped the breakpoints of a complex chromosomal rearrangement (CCR) 46,XY,t(7;8;11;13) in a patient with MBS, which revealed 41 clustered breakpoints with typical hallmarks of chromothripsis. Among 12 truncated protein-coding genes, SEMA3A is known to bind to the MBS-associated PLXND1. Intriguingly, the CCR also truncated PIK3CG, which in silico interacts with REVL3 encoded by the other known MBS-gene REV3L, and with the SEMA3A/PLXND1 complex via FLT1. Additional studies of other complex rearrangements may reveal whether the multiple breakpoints in germline chromothripsis may predispose to complex multigenic disorders. Keywords Moebius syndrome; chromothripsis; SEMA3A; SEMA3D; PIK3CG *Correspondence Lusine Nazaryan-Petersen, Blegdamsvej 3B, 2200, Copenhagen N, Denmark. [email protected]; Niels Tommerup, Blegdamsvej 3B, 2200, Copenhagen N, Denmark. [email protected]. DISCLOSURE DECLARATION The authors declare that they have no competing interests. Nazaryan-Petersen et al. Page 2 Moebius syndrome (MBS; MIM#157900) is a congenital disorder with malformations of Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author orofacial structures and the limbs, largely caused by unilateral or bilateral paralysis of the facial and abducens nerves (Kadakia et al., 2015). MBS is characterized by patients’ masklike facial expression caused by their inability to smile, frown or raise an eyebrow, which may lead to emotional or social adjustment issues (Broussard & Borazjani, 2008). The aetiology of MBS is not fully understood, however, genetic defects, abnormal vascular supply during embryogenesis and environmental toxic factors leading to abnormal brainstem development have been considered to be involved (D’Cruz et al., 1993; Kadakia et al., 2015; Verzijl et al., 2003). A genetic aetiology for MBS was suggested by several familiar cases with MBS, where both autosomal dominant (Kremer et al., 1996; Ziter et al., 1977) and recessive (Donahue et al., 1993) modes of inheritance have been described. Linkage analysis and chromosomal abnormalities have provided evidence for specific loci at 1p22; 3q21-q22; 10q21.3-q22.1 and 13q12.2–13, where several candidate genes have been proposed due to functional relevance (Supp. Table S1) (Kadakia et al., 2015). Sequencing studies have so far not identified mutations in specific genes at these loci (Uzumcu et al., 2009; B van der Zwaag et al., 2002; Bert van der Zwaag et al., 2004), but a recent study of 103 MBS patients revealed mutations in the genes PLXND1 (MIM# 604282) and REV3L (MIM# 602776) (Tomas-Roca et al., 2015). In addition, mutations in TUBB3 (MIM# 602661) have been identified in two patients with overlapping MBS symptoms (Nakamura et al.,, 2018; Patel et al., 2017). Here, using next generation mate-pair sequencing, we provide detailed genomic mapping and characterization of the breakpoint-junctions (BPJs) of a previously reported complex chromosomal rearrangement (CCR), 46,XY,t(7;8;11;13), (Borck et al., 2001). At that time, nine breakpoints were suggested: four located on 7q21.1–7q36; two on 8q21.3; two on 11p14.3 and one on 13q21.2. In the original chromosome analysis in 1976, no report of parental mosacisim was noted. Therefore, we concluded that this CCR is a de novo event. The patient had typical symptoms of MBS, such as micrognathia and congenital paresis of the facial muscles resulting in sucking and swallowing difficulties. In addition, gynecomastia was noted in the clinical description but it is unknown whether he had micropenis, delayed puberty, hypo-/anosmia or other manifestations related to hypogonadotropic hypogonadism. He had severe intellectual disability with limited language. Hearing loss was noticed from the age of 50 years. He died 54 years old. The patient’s parents and two siblings were healthy. The study has been approved by the Danish Data Protection Agency (2012–54-0053) and written consent was obtained from the brother of the patient. Mate-pair libraries were prepared using Nextera mate-pair kit following the manufacturers’ instructions (Illumina, San Diego, CA, USA) and. the final library was subjected to 2x100 bases paired-end sequencing on an Illumina HiSeq2500 sequencing platform. FASTQ files were aligned to GRCh37 (hg19) using BWA-mem (Li, 2013). Only SVs with at least five confirming read-pairs were considered. Sample-specific SVs were identified by filtering the predicted SVs against DGV (Database of Genomic Variants, http:// dgv.tcag.ca/dgv/app/home) and against an in-house database. The BPs indicated by MPS analysis were further validated by PCR and Sanger sequencing using standard procedures (primers and PCR conditions are provided in Supp Table S3) (Nazaryan et al., 2014). The BPJ sequences were split up at the breakpoint and aligned to genomic DNA of the Hum Mutat. Author manuscript; available in PMC 2020 August 01. Nazaryan-Petersen et al. Page 3 breakpoint region to visualize indels, microhomology, insertions and repeat elements within Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author the breakpoint. We used these molecular signatures at the BPJs to infer to the underlying mutational and repair mechanisms, e.g. non-homologous end-joining (NHEJ) (Lieber, 2010); microhomology-mediated end-joining (MMEJ) (McVey & Lee, 2008). The inserted L1M2 element at the BPJ 7–15_7–13 was checked for a possible underlying retrotransposition mechanism (Nazaryan-Petersen et al., 2016).. The identified truncated protein coding genes and topological associated domains (TADs) (Dixon et al., 2012; Rao et al., 2014) defined for human IMR90 fibroblasts (Supp. Table S2) at the breakpoints were analyzed for possible overlaps with known and candidate MBS genes (Supp. Table S1). The fused truncated genes with the same transcriptional orientation were evaluated in silico for the presence of open reading frames using the ExPASy Translate tool (http://www.expasy.org). We also performed in silico protein-protein interaction (PPI) analysis of the truncated genes/TADs and known and candidate MBS genes from the literature (Supp. Table S1) by using the STRING online database (Szklarczyk et al., 2017; https://string-db.org/) with a minimum required interaction score of 0.400. By mate-pair sequencing, we identified 41 breakpoints in total (Supp. Figure S1A, Supp. Table S4). As seen on the circos plot (Supp. Figure S1C), the breakpoints are clustered within relatively small genomic regions, involving only single chromosomal arms (7q; 8q and 11p), typical of chromothripsis. No major imbalances were detected using the depth of coverage of the mate-pair reads. Intra- and interchromosomal structural variants were indicated by 41 BPJs, including 15 translocations, 11 inversions, 7 duplication-type and 8 deletion-type of rearrangements (Supp. Figure S1A, S1C, Supp. Table S5), which have been reported to the Database of Genomic Structural Variation (dbVAR, accession number: nstd161, https://www.ncbi.nlm.nih.gov/dbvar/studies/nstd161/). Based on our NGS data, we paired the chromosomal fragments together by order and orientation to establish the derivative chromosomes (Supp. Figure S1B). At the mate-pair sequencing level the size of the identified fragments varied from ~2.9 kb to ~23.3 Mb. We confirmed 39 of the 41 BPJs (95.1%) by PCR and Sanger